ENHANCEMENT OF SOMATIC EMBRYOGENESIS IN THEOBROMA CACAO USING PHTYOCYANIN-LIKE-1 ARABINOGALACTAN (PLA) PROTEIN DOMAINS
Open Access
- Author:
- Lai, Tina
- Graduate Program:
- Plant Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- September 27, 2016
- Committee Members:
- Wayne Roger Curtis, Thesis Advisor/Co-Advisor
- Keywords:
- Phytocyanin-like-1
Arabinogalactan
AGP
PLA
PLA-domain
PLA protein
Plant Somatic Embryogenesis
In Vitro Plant Propagation - Abstract:
- PLA1, also known as Phytocyanin-Like-Arabinogalactan-1 is a protein domain that has been shown to improve somatic embryogenesis (SE) in cotton (GhPLA1). Poon et al. demonstrated that when GhPLA1 was added to tissue culture media, it increased the production of embryogenic calli by 2.68 fold compared to non-protein treated controls. These results inspired this thesis research to develop PLA protein as a potential purified tissue culture additive and to characterize its function of SE enhancement in the recalcitrant Theobroma cacao tissue culture system. Two PLA orthologs were identified from the Theobroma cacao genome based on 1) gene homology and 2) microarray expression data (TcPLA1 and TcPLA3). These two genes were cloned into pET14b, an Escherichia coli protein expression vector. TcPLA1 and TcPLA3 proteins were expressed from E. coli and isolated using Ni-Resin HIS-tag purification. GhPLA1 in addition to TcPLA1 and TcPLA3 proteins were applied to T. cacao tissue culture system via a protein solution drip method described in detail in Chapter 5. Specifically, the goal of this thesis was to evaluate the function of PLA proteins in T. cacao via 1) observed effects on embryo production and 2) effects of protein treatment on SE transcription factor genes such as BBM, LEC1, LEC2, AGL15, and FUS3. Embryo production was observed after 2 months of protein treatment. SE gene expression was observed via RT-qPCR. Results show GhPLA1 protein treatment increased embryo production and enhanced SE gene expression of transcription factor AGL15. Understanding how PLA interacts with SE genes can give insight into the complex orchestration of gene expression during plant embryo development. There were many challenges in completing this thesis work and unfortunately there was not enough time to thoroughly evaluate TcPLA1 and TcPLA3 protein treatment. Although, preliminary results of TcPLA1 and TcPLA3 protein treatment in T. cacao tissue culture are promising. Results show TcPLA1 and TcPLA3 induced more embryogenic responsive explants compared to no protein treated negative controls. PLA proteins may prove to be a valuable addition to the in vitro toolbox (beyond plant hormones) for plant species that are difficult to transform or propagate.